Treatment method and product for uterine fibroids using purified collagenase

ABSTRACT

The invention relates to compositions and methods for treating uterine fibroids, wherein a uterine fibroid treatment agent comprising collagenase in an amount effective to cause shrinkage of uterine fibroids is injected or inserted into the uterine fibroid.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.No. 61/790,070, filed on Mar. 15, 2013, which is hereby incorporated byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates to methods and products for medicaltreatment designed to reduce, shrink change the viscoelastic propertiesof, soften or eliminate unwanted tissue such as uterine fibroid tissue.

BACKGROUND OF THE INVENTION

Uterine fibroid tumors (also referred to as “uterine fibroids” or“leiomyomas”) are non-cancerous tumors of the uterine wall that occur in20 to 50% of women, and have an astonishingly high accumulativeincidence. Current studies demonstrate that by age 50, 70-80% of womenhave developed uterine fibroids, with higher incidence inAfrican-American women, who commonly develop fibroids earlier than otherracial groups. A significant number of those with uterine fibroidssuffer from debilitating pelvic pain, heavy and prolonged bleeding(which may lead to anemia and iron deficiency), bowel and bladderdysfunction and infertility. Uterine fibroids also cause symptoms suchas low back pain, urinary frequency and urgency, pain during intercourse(dyspareunia), and negative impact on fertility. They are associatedwith high morbidity from uterine bleeding and pain along with healthcare costs estimated to be between $2.1 and $34.4 billion annually inthe United States alone. Therefore, uterine fibroids have a significantimpact on the health and well-being of reproductive age women and on theeconomy. After menopause, generally, fibroids shrink and only rarelycause problematic symptoms.

The etiology of this disease remains unknown, therefore there are nomethods of preventing uterine fibroids. Several treatments areavailable, but hysterectomy is the only treatment which can permanentlyeliminate fibroids. The majority of the hysterectomies performed in theUnited States each year are due to uterine fibroids. It is obvious, butrarely stated in the literature, that hysterectomies lead to irrevocableloss of fertility. This invasive surgery also has a high cost,financially, socially and otherwise. It is associated with lengthyrecovery times, potential for sometimes severe postoperativecomplications, and physical discomfort. Thus, this solution is far fromideal.

Other surgical methods such as myomectomy (surgical removal of thefibroid tissue leaving the remainder of the uterus intact) is commonlyused, but may not be suitable in cases where the fibroids are too largeor too numerous to leave enough normal tissue behind. Further, thefibroids often recur. In addition, about three-quarters of myomectomysurgeries are open surgeries involving an abdominal incision. Therefore,this method also is associated with complications, discomfort, longrecovery, and potentially loss of fertility as well. Myolysis andcryomyolysis, in which uterine fibroids are burned or frozen vialaparoscopic surgery, can be used to cause the fibroids to shrink anddie over time. However, multiple punctures of the fibroids are needed totreat the entire tumor, and the treatment may cause adhesionspost-surgery. MRI guided focused ultrasound also is used in thetreatment of uterine fibroids, but this procedure is very expensive, anddoes not permanently eliminate the fibroids. Uterine arteryembolization, during which a catheter is inserted into a femoral arteryand guided to a uterine fibroid artery for injection of small particlesinto the fibroid artery, blocks the supply of blood, resulting in deathof the fibroid tissue. Although this procedure is less invasive thantraditional surgery, post-surgical pain is a frequent problem. Inaddition, this therapy, like hysterectomy, is considered a standardtreatment for women with no desire for future fertility. Alternatively,MRgFUS provides noninvasive fibroid-specific therapy utilizinghigh-intensity ultrasonography through the abdominal wall to causecoagulative necrosis in specific fibroids. Guidance and thermalmonitoring is provided by dynamic real-time magnetic resonance imaging.The surgical procedures to destroy uterine fibroids while preserving theuterus also have major drawbacks and often are not completelysuccessful, due to re-growth of the fibroid tumors.

Non-surgical, pharmaceutical-based medical therapies are available.Fibroids often are treated by medications aimed at treating the symptomsrather than the fibroid tumors themselves. In the early stages,physicians employ a “wait-and-see” approach, with no treatment orsymptomatic treatment until the condition impacts the ability of thepatient to function in normal life. Most fibroids are not treated unlessthey are causing symptoms. However, even in the absence of hysterectomy,fibroids, particularly subserosal fibroids, also can lead toinfertility.

The pharmacotherapies which are aimed at shrinking fibroid tumors orpreventing increase in size have been disappointing and often havesignificant side effects. Drugs have been studied and sometimes areeffective at shrinking uterine fibroids, but many of these non-surgicaltherapies have been associated with systemic side effects and thereforehave not been approved for clinical use. For example, selectiveprogesterone receptor modulators (SPRM) have not been approved by theFDA due to their effects on the endometrium. Only one drug has beenapproved for use to shrink uterine fibroids: leuprolide acetate. Thisdrug is used as a short-term treatment which suppresses ovarian function(and therefore causes significant menopausal side effects), shrinkingfibroids prior to surgery. Other medical therapies have been suggestedin the recent past such as selective estrogen receptor modulators(SERM), but clinical trial results have been disappointing.

Current treatment options for uterine fibroids are inadequate. Hence,there is a continuing need in the art for alternative therapies for thetreatment of uterine fibroids which are not open procedures and whichpreserve the patient's uterus. In particular, because treatment ofuterine fibroids costs billions of health care dollars each year, andyet this condition remains a significant problem, there is a need fortreatment methods that reduce or eliminate symptoms, provide reliefwithout highly invasive procedures, and which preserve fertility.

SUMMARY OF THE INVENTION

The following brief summary is not intended to include all features andaspects of the present invention, nor does it imply that the inventionmust include all features and aspects discussed in this summary.

Embodiments of the invention are designed to provide the advantage offormulations, compositions and methods for treatment of uterine fibroidswhich do not require open surgical procedures and which preserve thepatient's uterus. Another advantage of the present invention is thatinjectable or insertable formulations are provided, which displayimproved retention of agents within uterine fibroid tissue, therebyimproving delivery efficiency, while at the same time minimizing adverseeffects such as nonspecific damage and systemic effects. Theseformulations, compositions and methods include injectable, implantableor insertable formulations which contain one or more uterine fibroidtreatment agents, preferably at least a purified collagenase in anamount effective to shrink or eliminate fibroids that are exposed to theformulation.

The foregoing and other objects, features and advantages of theinvention will be apparent from the following more particulardescription of preferred embodiments of the invention, as illustrated inthe accompanying drawings in which like reference characters refer tothe same parts throughout the different views. The drawings are notnecessarily to scale, emphasis instead being placed upon illustratingthe principles of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The application file contains at least one drawing executed in color.Copies of any patent or patent application publication from thisapplication containing color drawing(s) will be provided by the Officeupon request and payment of the necessary fee.

FIGS. 1A and 1B are electron micrographs (×31000) showing collagenfibrils in a uterine fibroid (1A) and in corresponding myometrium (1B).

FIG. 2 is a photograph of uterine tissue cubes.

FIGS. 3A and 3B are photographs of a uterine tissue cube undergoinginjection (FIG. 3A) and injected uterine tissue cubes undergoingincubation (FIG. 3B).

FIGS. 4A and 4B are photographs of an excised uterus, showing a uterinefibroid (FIG. 4A) and a uterine fibroid undergoing injection (FIG. 4B).

FIG. 5 is a pair of photographs showing fibroid tissue cubes injectedwith vehicle (control) or collagenase, after 48 hour incubation.

FIG. 6 is a set of four micrographs. FIGS. 6A and 6B show controltissue. FIGS. 6C and 6D show tissue that has been degraded withcollagenase.

FIG. 7 is a scanning electron micrograph of the BD 3D OPLA® scaffold.

FIG. 8 is a micrograph showing H&E stain of fibroid cells seeded onto anOPLA scaffold and cultured for 9 days (Zeiss Axio Imager® widefieldfluorescence microscopy).

FIG. 9 is a bright field image (9A) with fluorescent image overlay (9B)showing primary cultures of fibroid cells 8 days after static seeding(Zeiss Lumar® stereoscopic image, stained with fluorescein-phalloidinfor f-Actin).

FIG. 10 is a micrograph showing primary fibroid cells cultured on OPLAscaffolds and fixed in situ. Images 10A and 10B were taken from the samefield of vision every 10 micrometers.

FIG. 11 is a bar graph showing the percentages of the types of collagenin fibroids.

FIG. 12 is an SDS-PAGE analysis of collagen content in a fibroid sample(Nydea Aviles and Sergey Leikin, NIH) FIG. 12A shows total collagenunder non-reducing conditions. FIG. 12B shows total collagen underreducing conditions. FIG. 12C shows a sample depleted of type V collagenby selective salt precipitation. FIG. 12D shows a sample enriched intype V collagen by selective salt precipitation (Feng L, Leikin S et al.presented at XXIInd Meeting of the Federation of European ConnectiveTissue Societies (FECTS), Jul. 3-7, 2010).

FIG. 13 shows how TGF-β may be involved in fibrosis, which may beconsidered a form of altered tissue repair. Cell injury in myometrialcells leads to activation of repair. In fibroids, wound healing halts atthe proliferation stage, collagen accumulates. (Leppert et al. A newhypothesis about the origin of uterine fibroids based on gene expressionprofiling with microarrays. Am J Obstet Gyn 2006; 95: 415-20.)

FIG. 14 shows structural changes associated with uterine fibroids. Thechange in cell shape from smooth muscle to fibroid cell appears to be amyofibroblast-like cell, suggesting that mechanotransduction plays arole in fibroid formation. FIG. 14A shows fibroid cells with phalloidinstaining with magnification 40×. FIG. 14B shows phalloidin staining of asections from a matched myometrial sample with magnification 40×. FIG.14C shows fibroid specimen and magnification 21,000× and FIG. 14D showsa matched myometrium specimen at magnification 21,000×. Note the angularcell shape, reduced cytoplasm, and notched nucleus in the fibroid (C)compared with myometrium (D).

FIG. 15 is a schematic modified from FIG. 3 of Hinz and Gabbiani(Current Opinions in Biotechnology 2003, 14:38-46) that shows how TGF-βis a key mediator in fibrosis. TGF-β stimulates differentiation offibroblasts into myfibroblasts, promotes the transcription of collagen,and stimulates extracellular matrix (ECM) accumulation. The schematicalso shows the role of mechanotransduction and the role of the ECM infibroid development. The ECM accumulation exerts compression on cells.The cells product increased ECM, which leads to mechanotransduction ofincreased collagen synthesis and deposition. Apoptosis of fibroid cellsdoes not occur, and cells produce even more collagen.

FIG. 16 is a set of representative gels of products from reversetranscriptase-PCR reactions for decorin, TGF-β₁, TGF-β₃ andinterleukin-4. Paired leiomyoma (fibroid) and myometrium samples are asdepicted. Leiomyoma-myometrium pairs are arranged in 10-fold dilutionsof total RNA in each reaction. The negative control was a reactioncontaining primers but no RNA template. GAPDH was amplified as aninternal control for assessment of amplification and similar amounts ofRNA in samples. Lane markers are shown at the far left of the gel.(Catherino et al. (2004) Genes, Chromosomes & Cancer 40:204-217.)

FIG. 17 shows the complex shear modulus (stiffness) of human fibroidtissue treated with purified clostridial collagenase.

DETAILED DESCRIPTION OF THE INVENTION

Collagen is the major structural constituent of mammalian organisms andmakes up a large portion of the total protein content of skin and otherparts of the animal body. Various skin traumas such as burns, surgery,infection and accident are often characterized by the erraticaccumulation of fibrous tissue rich in collagen and having increasedproteoglycan content. In addition to the replacement of the normaltissue which has been damaged or destroyed, excessive and disfiguringdeposits of new tissue sometimes form during the healing process. Somediseases and conditions are associated with excess collagen depositionand the erratic accumulation of fibrous tissue rich in collagen. Suchdiseases and conditions are collectively referred to herein as“collagen-mediated diseases”.

It has now been found that uterine fibroids are a collagen-mediateddisease, associated with excess collagen deposition and the erraticaccumulation of fibrous tissue rich in collagen. The considerablevariation in growth rates over time of individual fibroids, andmicroarray studies revealing that genes encoding for ECM proteins orrelated to ECM synthesis and secretion account for a large portion ofchanges in gene expression in fibroids compared with myometrium makedysregulation of ECM (extracellular matrix) a possible contributingfactor to this condition.

Transforming growth factor (TGF) plays a role in fibroid development.Fibroids grow by deposition of altered collagen. The expression of othermolecules is likewise altered in fibroids. For example, dermatopontinexpression is decreased, fibronectin and glycosaminoglycans (GAG) areincreased, alpha 11 integrin, a collagen-binding integrin is expressed.In addition, fibroids are resistant to apoptosis.

Recent studies indicate that fibroids are formed by the accumulation ofextracellular matrix (ECM) as well as by cellular proliferation. SeeFIG. 1, noting the disordered collagen fibrils in the fibroid tissue.The appearance and spatial orientation of collagen fibrils in uterinefibroids were shorter, randomly aligned and widely dispersed comparedwith those of the myometrium. They were non-aligned and not parallelwhereas in the adjacent myometrium the fibrils were well packed andparallel in orientation to each other, a finding that is characteristicof collagen containing tissue. Myofibroblast type cells (elongatedappearance, notched nucleus) also have been found in uterine fibroids.The notched appearance of the fibroid cell nucleus represents foldingand envaginations of the nuclear membrane due to cell contraction bystress fibers.

Therefore, the present invention takes advantage of collagenase, anenzyme that has the specific ability to digest collagen, to treatuterine fibroids. Degradation of the collagen not only causescollagenolysis, it also reduces the increased cell compression leadingto mechanotransduction. Thereby, the cycle of increased collagensecretion and enlargement of the uterine fibroid is broken. In summary,uterine fibroids contain an abundance of altered collagen consistentwith fibrosis and stiffness. A stiff extracellular matrix (ECM) exertsforce against individual cells. Mechanotransduction alters cellsignaling and prevents apoptosis, and thus collagen accumulationcontinues. (See, FIG. 15.) Uterine fibroids grow at individual ratessuggesting that mechanical transduction of tumors is responsible forvariation in growth rates.

This specification describes embodiments of an invention for treatmentto reduce the symptoms of uterine fibroids, shrink uterine fibroids,reduce the stiffness and mechanical stress of fibroid tissue on theuterus and/or eliminate uterine fibroids by local delivery of a purifiedcollagenase composition to avoid systemic side-effects and harm to othertissues. In general, some of the preferred methods use a syringe andneedle under ultrasound or other visualization for guided injection ofpurified collagenase directly into the uterine fibroid tissue to betreated. The collagenase product preferably is in a vehicle fordelivery, such as a nanocarrier or other protective or sustained releasecarrier.

Because the center of fibroids is more fibrotic and contains smallervascular capillary beds than the periphery, and due to a dense vascularcapsule which surrounds the fibroid tumor, systemic therapy is notlikely to provide therapeutic tissue levels of a drug in the fibroidcenter while leaving the likely possibility of systemic effects. Thus,pharmacotherapy has not been successful for uterine fibroids. The localinjection of a treatment agent under imaging guidance allows for exacttissue placement of the drug and greatly reduces the chance of systemiceffects.

Uterine fibroids are classified into several types, based on theirlocation, including subserosal, intramural, submucosal, pedunculatedsubmucosal, fibroid in statu nascendi, and fibroid of the broadligament. Any and all of these uterine fibroids are contemplated fortreatment using the invention.

Myometrial Hyperplasia is a condition which can mimic uterine fibroidsymptoms and may be a precursor lesion of these tumors. It is structuralvariation with irregular zones of hypercellularity and increasednucleus/cell ratio, causing a bulging, firm, enlarged uterus. Thecondition often leads to hysterectomy. Deeper MMH has lower cellularity,and tends to have increased collagen. Therefore, this condition also maybe treated using the methods and compositions of the invention.

The local treatment of uterine fibroids by injection of collagenase canbe conducted in an office or clinic visit under ultrasound guidance withminimal chance for sequelae. This method can be used to treat small tomoderate size fibroids or asymptomatic fibroids, which currently are nottreated at all, allowing the clinician to prevent potentiallydebilitating symptoms and preservation of fertility in women ofchild-bearing years, and also larger fibroids, eliminating the need forhysterectomy for this disease. Thus, the methods of this invention arecontemplated to be useful to treat any stage or type of uterine fibroiddisease.

Collagenase for use according to the invention may be obtained from anyconvenient source, including mammalian (e.g., human, porcine),crustacean (e.g., crab, shrimp), fungal, and bacterial (e.g., from thefermentation of Clostridium, Streptomyces, Pseudomonas, Vibrio orAchromobacter iophagus). Collagenase can be isolated from a naturalsource or can be genetically engineered/recombinant. One common sourceof crude collagenase is from a bacterial fermentation process,specifically the fermentation of Clostridium histolyticum. The crudecollagenase obtained from C. histolyticum can be purified using any of anumber of techniques known in the art of protein purification, includingchromatographic techniques. Collagenase compositions useful for theinvention also can be prepared using any commercially available orisolated collagenase activity, or by mixing such activities. Forexample, purified collagenase can be provided by BiospecificsTechnologies, Lynbrook, N.Y.

Preferred collagenases for use in the invention are from C.histolyticum, i.e., collagenase class I and class II. A practicaladvantage of using C. histolyticum for the production of collagenases isthat it can be cultured in large quantities in simple liquid media, andit regularly produces amounts of proteolytic enzymes which are secretedinto the culture medium. Bovine products have been used in culture mediain the fermentation of C. histolyticum, but these run the risk ofcontamination by agents which cause transmissible spongiformencephalopathies (TSEs; e.g., prions associated with bovine spongiformencephalopathy or “mad cow disease”). Therefore, it is preferred toavoid such bovine products. An animal-product-free system is preferred.The H4 strain of Clostridium histolyticum, originally developed in 1956can serve as a source for cells for culture. This strain, and a strainderived from the H4 strain, named the ABC Clostridium histolyticummaster cell bank (deposited as ATCC 21000) were developed using animalproducts, but are suitable to use in the invention.

U.S. Pat. No. 7,811,560, which is incorporated herein by reference inits entirety, discloses methods of producing collagenases. Using soybeanderived fermentation medium, the methods described therein generatedseparately highly purified collagenase I and II. This patent alsodiscloses methods of producing highly purified collagenases usingculture media containing porcine-derived products. Any of these methodsare suitable for use with the invention. U.S. Patent Publication2010/0086971, which is also incorporated herein by reference in itsentirety, discloses numerous fermentation recipes which are based onvegetable peptone, including soybean-derived peptone, orvegetable-derived peptone plus fish gelatin. The methods described inthis publication are suitable to produce growth of Clostridium andcollagenase activities. These methods also are suitable and contemplatedfor use with the invention, however any method known in the art ofproducing collagenase enzyme activity may be used.

In preferred culture methods, the peptone is from a plant sourceselected from the group consisting of soy bean, broad bean, pea, potato,and a mixture thereof. The peptone may be selected from the groupconsisting of Oxoid VG100 Vegetable peptone No. 1 from pea (VG100),Oxoid VG200 Vegetable peptone phosphate broth from Pea (VG200), MerckTSB CASO-Bouillion animal-free (TSB), Invitrogen Soy bean peptone No 110papainic digest (SP6), Fluka Broad bean peptone (BP), OrganotechniePlant peptone E1 from potato (E1P), BBL Phytone™ peptone and BD DifcoSelect Phytone™.

In a preferred embodiment of the invention, a single type of peptone ispresent in the nutrient composition of the invention, whereby thepeptone is selected from the group consisting of BP, E1P, Soy beanpeptone E110, VG100, and VG200, and whereby the concentration of thepeptone in the composition is about 5% weight by volume. In yet anothervery much preferred embodiment of the invention, a single type ofpeptone is present in the nutrient composition of the invention, wherebythe peptone is BBL phytone peptone or Difco Select Phytone™ UF, andwhereby the concentration of the peptone in the composition is about10-13% weight by volume.

Preferred methods of isolating collagenase avoid undesirablecontaminating proteases such as clostripain. Clostripain, a cysteineprotease, is believed to be a major cause of collagenase degradation andinstability, and is present in Clostridium culture. When such proteasesare present in a crude collagenase mixture, one must take extraprecautions to neutralize the proteases, including using proteaseinhibitors, such as leupeptin, and performing all of the purificationsteps in specially designed cold rooms with chilled solutions to reduceprotease activity. Preferred methods of isolation therefore takeadvantage of one of two approaches to avoid clostripain: removeclostripain as early as possible in the purification method or reduceclostripain production during the fermentation stage.

Preferred collagenase compositions are produced by fermenting C.histolyticum in medium free of animal material-derived ingredients andare substantially free of clostripain, and thus are highly stable.“Substantially free” indicates that the collagenase contains less than10 U clostripain per mg total collagenase, more preferably less than 5U/mg, and most preferably about 1 U/mg or less, and/or that no visibleband appears representing clostripain and/or degraded collagenase onSDS-PAGE gel compared to a reference standard.

Preferred methods for purifying collagenase involve using a “lowglucose” medium as described herein, which contains less than about 5g/L glucose, more preferably less than about 1 g/L, even more preferablyless than about 0.5 g/L glucose, or is glucose-free, for culture of C.histolyticum. High salt concentrations in the growth media can reducethe amount of clostripain produced in culture, thus preferred media forC. histolyticum culture contain greater than about 5 g/L (or 0.5% w/v)total salt, preferably greater than about 7.5 g/L (or 7.5%) total salt,and more preferably about 9 g/L (or 9%) or more. It is contemplated thatany salt known to be suitable for use in microbiological fermentationmedia may be used in the current invention. In a preferred embodiment,chloride, phosphate or sulfate salts may be used. In a more preferredembodiment, the salts may be sodium chloride, potassium chloride,monosodium phosphate, disodium phosphate, tribasic sodium phosphate,potassium monophosphate, potassium diphosphate, tripotassium phosphate,calcium chloride, magnesium sulfate or various combinations thereof. Incertain embodiments, potassium diphosphate may be about 0.1-0.3%,potassium phosphate may be about 0.75% to 0.175%, sodium phosphate maybe about 0.2-0.5%, and/or sodium chloride may be about 0.15-0.35%.Preferably, the medium further comprises magnesium sulfate and vitamins,including, riboflavin, niacin, calcium pantothenate, pimelic acid,pyridoxine and thiamine.

In another preferred embodiment, the nutrient composition may contain0.5-5% yeast extract, more preferably about 1-4%, and most preferablyabout 1.5-2.5%. Yeast extract is available from a variety of suppliers,including Cole Parmer (Vernon Hills, Ill.) and Fisher Scientific(Pittsburgh, Pa.).

In yet a preferred embodiment of the invention, the pH of the media isbetween pH 7 and pH 8. Even more preferred is a pH between about pH 7.2and about pH 7.7, most preferably about 7.4.

The collagenase contemplated for use with the invention can be anycollagenase which is active under the necessary conditions. However,preferred compositions contain a mass ratio of collagenase I andcollagenase II which is modified or optimized to produce a desired oreven a maximal synergistic effect. Preferably, collagenase I andcollagenase II are purified separately from the crude collagenasemixture produced in culture, and the collagenase I and collagenase IIare recombined in an optimized fixed mass ratio. Preferred embodimentscontain a collagenase I to collagenase II mass ratio of about 0.5 to1.5, more preferably 0.6 to 1.3, even more preferably 0.8 to 1.2, andmost preferably, 1 to 1, however any combination or any singlecollagenase activity may be used.

A preferred method of producing collagenase which is contemplated foruse with the invention involves fermenting C. histolyticum in anon-mammalian or non-animal medium, wherein the culture supernatant issubstantially clostripain-free. The collagenases so produced can beisolated, purified, and combined to provide a composition for use in theinvention which comprises a mixture of collagenase I and collagenase IIin an optimized fixed mass ratio which is substantiallyclostripain-free. The crude collagenase obtained from fermentation of C.histolyticum may be purified by a variety of methods known to thoseskilled in the art, including dye ligand affinity chromatography,heparin affinity chromatography, ammonium sulfate precipitation,hydroxylapatite chromatography, size exclusion chromatography, ionexchange chromatography, and/or metal chelation chromatography.Additionally, purification methods for collagenases are known, such as,for example, those described in U.S. Pat. No. 7,811,560, which is herebyincorporated by reference in its entirety.

Both collagenase I and collagenase II are metalloproteases and requiretightly bound zinc and loosely bound calcium for their. Bothcollagenases have broad specificity toward all types of collagen.Collagenase I and Collagenase II digest collagen by hydrolyzing thetriple-helical region of collagen under physiological conditions. Eachcollagenase shows different specificity (e.g. each have a differentpreferred target amino sequence for cleavage), and together they havesynergistic activity toward collagen. Collagenase II has a higheractivity towards all kinds of synthetic peptide substrates thancollagenase I as reported for class II and class I collagenase in theliteratures.

The preferred collagenase consists of two microbial collagenases,referred to as Collagenase ABC I and Collagenase ABC II. The terms“Collagenase I”, “ABC I”, and “collagenase ABC I” mean the same and canbe used interchangeably. Similarly, the terms “Collagenase II”, “ABCII”, and “collagenase ABC II” refer to the same enzyme and can also beused interchangeably. These collagenases are secreted by bacterialcells. Preferably, they are isolated and purified from Clostridiumhistolyticum culture supernatant by chromatographic methods. Bothcollagenases are special proteases and share the same EC number (E.C3.4.24.3). However, a collagenase or a combination of collagenases fromother sources are contemplated for use with the invention. CollagenaseABC I has a single polypeptide chain consisting of approximately 1000amino acids with a molecular weight of 115 kDa. Collagenase ABC II hasalso a single polypeptide chain consisting of about 1000 amino acidswith a molecular weight of 110 kDa.

Collagenase acts by hydrolyzing the peptide bond between Gly-Pro-X,wherein X is often proline or hydroxyproline. Collagenase I acts at lociat ends of triple-helical domains, whereas Collagenase II cleavesinternally. Hydrolysis continues over time until all bonds are cleaved.

Preferably, the collagenase product is at least 95% pure collagenase(s)and is substantially free of any contaminating proteases. Morepreferably, the collagenase product is 97% pure and most preferably 98%pure or more as determined by one or more of the following: sodiumdodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE); highperformance liquid chromatography (HPLC); reverse-phase HPLC; or byenzymatic assays. The preferred collagenase product is essentiallyclostripain-free, and the purification preferably is performed in theabsence of leupeptin. The preferred collagenase product for use with theinvention has at least one specification selected from Table 1 below.

TABLE 1 Preferred Specifications for Collagenase Products SpecificationTest ABC-I ABC-II Appearance Clear colorless and essentially free fromparticulate matter Endotoxin <10 EU/mL Identity (and purity) by Majorcollagenase Major collagenase SDS-PAGE (Reduced band between 98- bandbetween 97- conditions, Coomasie) 188 kDa 200 kDa ≥95% ≥95% SRC assay(ABC-I) 1967-3327 SRC NA units/mg GPA assay (ABC-II) NA81934-119522 GPAunits/mg Analysis of Proteins ≥98% main peak; ≤2% aggregates by areaHPLC System (Aggregation by size exclusion chromatography) Identity andpurity by Major peak (ABC I or ABC II), ≥95% by reverse phase liquidarea; Retention times of ABC-I and ABC-II chromatography) within 5% ofreference Clostripain assay (BAEE ≤1 U/mg  assay) Bioburden  <1 cfu/mL

The collagenase products described for use herein are useful for thetreatment of collagen-mediated disease, including uterine fibroids.Examples of other collagen mediated-diseases that may be treated by thecompositions of the invention include but are not limited to:Dupuytren's disease; Peyronie's disease; frozen shoulder (adhesivecapsulitis), keloids; tennis elbow (lateral epicondylitis); scarredtendon; glaucoma; herniated discs; adjunct to vitrectomy; hypertrophicscars; depressed scars such as those resulting from inflammatory acne;post-surgical adhesions; acne vulgaris; lipomas, and disfiguringconditions such as wrinkling, cellulite formation and neoplasticfibrosis.

In addition to its use in treating specific collagen-mediated diseases,the compositions of the invention also are useful for the dissociationof tissue into individual cells and cell clusters as is useful in a widevariety of laboratory, diagnostic and therapeutic applications. Theseapplications involve the isolation of many types of cells for varioususes, including microvascular endothelial cells for small diametersynthetic vascular graft seeding, hepatocytes for gene therapy, drugtoxicology screening and extracorporeal liver assist devices,chondrocytes for cartilage regeneration, and islets of Langerhans forthe treatment of insulin-dependent diabetes mellitus. Enzyme treatmentworks to fragment extracellular matrix proteins and proteins whichmaintain cell-to-cell contact. In general, the compositions of thepresent invention are useful for any application where the removal ofcells or the modification of an extracellular matrix, are desired.

The collagenase compositions according this invention are designed toadminister to a patient in need thereof a therapeutically effectiveamount of a collagenase composition as described, or a therapeuticallyeffective amount of a pharmaceutical collagenase formulation asdescribed. A “therapeutically effective amount” of a compound,composition or formulation is an amount of the compound which confers atherapeutic effect on the treated subject, at a reasonable benefit/riskratio applicable to any medical treatment. A therapeutic effect includesbut is not limited to a shrinkage or reduction in the size of one ormore uterine fibroids (including elimination of the fibroid),liquification, partial liquification, or reduction in stiffness(increase in softness) or pressure in or around a uterine fibroid, achange in viscoelastic properties, or reduction in symptoms such aspain, hemorrhage and the like.

The therapeutic effect may be objective (i.e., measurable by some testor marker) or subjective (i.e., subject gives an indication of or feelsan effect), and may be determined by the clinician or by the patient.Effective doses will also vary depending on route of administration, aswell as the possibility of co-usage with other agents. It will beunderstood, however, that the total daily usage of the compositions ofthe present invention will be decided by the attending physician withinthe scope of sound medical judgment. The specific therapeuticallyeffective dose level for any particular patient will depend upon avariety of factors including the disorder being treated and the severityof the disorder; the activity of the specific compound employed; thespecific composition employed; the age, body weight, general health, anddiet of the patient; the time of administration, route ofadministration, and rate of excretion of the specific compound employed;the duration of the treatment; drugs used in combination orcontemporaneously with the specific compound employed; and like factorswell known in the medical arts.

The term “patient” or “patient in need” encompasses any mammal having auterus and uterine fibroids or symptoms thereof. Such “patients” or“patients in need” include humans or any mammal, including farm animalssuch as horses and pigs, companion animals such as dogs and cats, andexperimental animals such as mice, rats and rabbits. Preferred patientsare human females of child-bearing age.

The pharmaceutical compositions of this invention preferably areadministered by injection, insertion or implantation directly into oronto the uterine fibroid tissue to be treated, i.e. local administrationto the tissue to be treated. Other modes of administration contemplatedincluded, but are not limited to transvaginal instillation orapplication onto the affected tissues, instillation or applicationduring surgery (such as laparoscopy or hysteroscopy) onto the affectedtissues, i.e. topical administration to the fibroid tissue, by spray orother application of a liquid, fluid or gel formulation.

Formulations of the present invention are injected/inserted into uterinetissue in a variety of forms, by a variety of routes, using a variety ofapparatuses. In some embodiments, the formulation is injected/insertedusing an apparatus consisting of a simple needle (e.g., a 10 gauge orsmaller needle) and sample pusher (e.g., a mandrel or modifiedobturator). For example, according to one embodiment, a formulation(e.g., a rod-shaped or other shaped solid or semi-solid formulation,beads, suspension, gel, polymer or the like) is placed in the needle orin a syringe or other chamber affixed to the needle. Once the needle isplaced at the desired depth and location in the tissue, the pusher isused to push the sample from the needle and into the tissue. In someembodiments, the sample pusher is provided with a holding clip or it isprovided with a hollow end to secure the sample up to the time ofdelivery.

In still other embodiments, formulations in accordance with the presentinvention are injected/inserted via jet injection without a physicaldelivery channel such as a needle, as is known in the art. Typically, acompression system (e.g., a mechanical system or a gas, such as helium,nitrogen, carbon dioxide, etc.) is used to accelerate the formulationsto a high enough velocity so that the formulation can penetrate thetissue to a desired depth. Jet injector devices can be, for example,disposable, or reusable with medication cartridges that are prefilled ornon-prefilled medication cartridges. Examples of jet injectors includeBiojector® from Bioject, N.J., USA and the PowderJect® System fromPowderJect, UK. In other embodiments, a device is employed that coresout a section of the fibroid (e.g., a biopsy device or tissuemorcellator or laser radiation), thereby leaving behind a void forinsertion of a dosage form.

The formulations for collagenase delivery to a patient generally arecontemplated to comprise injectable or implantable formulations, or anyfluid, liquid, solid, semi-solid, gel, or other composition which issuitable to administer the collagenase to the tissue to be treated asdescribed herein. Formulations in accordance with the present inventionmay be formulated by any method known in the pharmaceutical arts. Thus,any injectable or implantable formulation known in the art andconsistent with collagenase activity may be used. Formulations whichcreate a depot or extended release of the active collagenase agent arecontemplated. In particular, injectable extended or sustained releasecompositions are preferred, however any implantable formulation can beused. Such compositions produce or form a depot effect, where activeagent is present in the tissue where administered and release activeagent over a period of time to continuously treat the tissue. Immediaterelease injectable formulations, where the active agent is immediatelyreleased for activity upon administration, also are contemplated for usewith the invention. These formulations are known in the art and can beadapted for use with the present invention by any person of skill.

In some embodiments, the injectable or insertable formulations of thepresent invention are solids, semi-solids or high-viscosity fluids. Thisimproves dosage retention in the tissue, thereby improving deliveryefficiency of the treatment agents and/or minimizing the adverse effectssuch as unintended, nonspecific tissue damage. “High viscosity” andother such terms are used herein to describe fluids having viscositiesgreater than 1000 centipoise as measured by any of a number of standardtechniques, including, for example, a Brookfield Kinematic Viscometer,model HBDV11+CP with a CPE-40 cone spindle, set at 37° C. and using a0.5 rpm speed setting. “Low viscosity” fluids have viscosities less thanthis value.

In some embodiments, a formulation in accordance with the presentinvention is injected into a patient in a fluid state, whereupon itconverts (or is converted) in vivo into a more readily retained form,for example, into a solid form (including conversion of an injectedliquid into a solid, conversion of an injected semi-solid into a solidand conversion of a liquid into a gel), into a semi-solid form(including conversion of an injected liquid into a semi-solid,conversion of an injected semi-solid into a semi-solid having increasedyield stress and/or viscosity and conversion of a liquid into a gel), orinto a high-viscosity fluid (including conversion of a low-viscosityfluid into a high-viscosity fluid, and conversion of a high-viscosityfluid into a higher-viscosity fluid).

Preferred formulations for injection into a uterine fibroid use acarrier or nanocarrier. Appropriate carriers include solid or semi-solidpellets, beads or gel-forming polymers, high-viscosity liquids and thelike to maintain the active collagenase in the tissue, protecting theactive enzyme from action of the tissue or tissue components which couldinactivate the collagenase, and allow steady release of the enzyme tothe tissue for treatment. Any injectable dosage form which can protectand contain the active compound(s) in place may be used. In mammals, C.histolyticum collagenase is inhibited rapidly in the blood stream byserum. Therefore, systemic administration, or administration underconditions where the collagenase can be deactivated, or orally, wherethe collagenase can be degraded by digestive enzymes, is problematic.

Nanocarriers are designed to deliver and protect drug therapeutics (e.g.proteins, for example) from degradation. A nanocarrier formulation alsois preferred because this method impedes diffusion and distribution ofthe drug away from the injected fibroid, prolongs release, delaysinactivation, and therefore reduces the frequency of repeat injections.Any such nanocarrier known in the art can be used with the invention.Some of these nanocarriers also are referred to as thermoresponsivedelivery systems.

Atrigel® comprises a water-insoluble biodegradable polymer (e.g.,poly(lactic-co-glycolic acid, PLGA) dissolved in a bio-compatible,water-miscible organic solvent (e.g., N-methyl-2-pyrrolidone, NMP). Inuse, collagenase is added to form a solution or suspension. Both thePLGA molecular weight and lactide-glycolide molar ratio (L:G ratio)governs drug delivery. Using an L:G ratio of from 50:50 to 85:15 and apolymer concentration of from 34 to 50%, clinical studies havedemonstrated a depot which was maintained for more than 3 months.

ReGel® is a 4000 Da triblock copolymer formed from PLGA and polyethyleneglycol (PEG, 1000 Da or 1450 Da) in repetitions of PLGA-PEG-PLGA orPEG-PLGA-PEG. ReGel® is formulated as a 23 wt % copolymer solution inaqueous media. A drug is added to the solution and upon temperatureelevation to 37° C. the whole system gels. Degradation of ReGel® tofinal products of lactic acid, glycolic acid and PEG occurs over 1-6weeks depending on copolymer molar composition. Chemically distinctdrugs like porcine growth hormone and glucagon-like peptide-1 (GLP-1)may be incorporated, one at a time, and released from ReGel®.

LiquoGel™ can work by mechanistically independent drug delivery routes:entrapment and covalent linkage. Two or more drugs can be delivered tothe tumor site using this carrier. LiquoGel™ is a tetrameric copolymerof thermogelling N-isopropylacrylamide; biodegrading macromer ofpoly(lactic acid) and 2-hydroxyethyl methacrylate; hydrophilic acrylicacid (to maintain solubility of decomposition products); andmulti-functional hyperbranched polyglycerol to covalently attach drugs.LiquoGel™ generally is formulated as a 16.9 wt % copolymer solution inaqueous media. The solution gels under physiological conditions anddegrades to release drug contents within 1-6 days.

Any of the above carriers can be used as a nanocarrier with theinvention. A preferred nanocarrier, however, contains hyperbranchedpolyglycerols (HPG), which have many desirable features. HPGs grow byimperfect generations of branched units and are produced in a convenientsingle step reaction. Previous problems of large polydispersities inmolecular weight in their production have been overcome. The resultingpolymers contain a large number of modifiable surface functional groupsas well as internal cavities for drug interaction. Other polymerapproaches cannot easily provide these properties without significantincreases in the number of synthetic steps and, consequently, cost. HPGpolymers are based on glycerol and because of structural similarity withpolyethylene glycol, is biocompatible.

Additional components optionally can be added to the polymer, therefore,modified HPG polymers and co-polymers of HPG are contemplated. Theseadditional components or monomers can include, for example, crosslinks,biodegradable moieties, and thermoresponsive moieties. For example,thermally responsive hydrogels are attractive for injection therapysince it is possible to inject the necessary fluid volume from a syringemaintained below body temperature and upon warming, the mechanicalproperties are increased, thereby restraining the material at theinjection site. Poly(N-isopropylacrylamide) (poly-NIPAAm) is a thermallyresponsive polymer with a lower critical solution temperature (LCST) ofapproximately 32° C. Copolymers of HPG with NIPAAm are thereforecontemplated for use with the invention, and are preferred. Thisnanocarrier has a versatile mesh size and can be customized to entrapsmall drug molecules, large proteins, or a mixture of components, andgels at body temperature to permit slow release as the nanocarrierbiodegrades.

In preferred embodiments of the invention, formulations exist as aliquid at temperatures below body temperature and as a gel at bodytemperature. The temperature at which a transition from liquid to geloccurs is sometimes referred to as the LCST, and it can be a smalltemperature range as opposed to a specific temperature. Materialspossessing an LCST are referred to as LCST materials. Typical LCST's forthe practice of the present invention range, for example, from 10 to 37°C. As a result, a formulation injected below the LCST warms within thebody to a temperature that is at or above the LCST, thereby undergoing atransition from a liquid to a gel.

Suitable LCST materials for use with the invention includepolyoxyethylene-polyoxypropylene (PEO-PPO) block copolymers. Twoacceptable compounds are Pluronic acid F127 and F108, which are PEO-PPOblock copolymers with molecular weights of 12,600 and 14,600,respectively. Each of these compounds is available from BASF (MountOlive, N.J.). Pluronic acid F108 at 20-28% concentration concentration,in phosphate buffered Saline (PBS) is an example of a suitable LCSTmaterial. One beneficial preparation is 22.5% Pluronic acid F108 in PBS.A preparation of 22% Pluronic acid F108 in PBS has an LCST of 37° C.Pluronic acid F127 at 20-35% concentration in PBS is another example ofa suitable LCST material. A preparation of 20% Pluronic acid F127 in PBShas an LCST of 37° C. Typical molecular weights are between 5,000 and25,000, and, for the two specific compounds identified above are 12,600and 14,600. More generally, materials, including other PEO-PPO blockcopolymers, which are biodisintegrable, and which exist as a gel at bodytemperature and as a liquid below body temperature can also be usedaccording to the present invention. Further information regarding LCSTmaterials can be found in U.S. Pat. No. 6,565,530 B2 and U.S. Pat. No.6,544,227 B2, each of which is hereby incorporated by reference.

Pharmaceutical formulations of the collagenase compounds for theinvention include a collagenase composition formulated together with oneor more pharmaceutically acceptable vehicles or excipients. As usedherein, the term “pharmaceutically acceptable carrier or excipient”means a non-toxic, inert, solid, semisolid or liquid filler, diluent,encapsulating material, vehicle, solvent, or formulation auxiliary ofany type, and may be made available in individual dosage forms or inbulk. Other dosage forms designed to create a depot of the activecompound also are contemplated for use with the invention. Dosage formsfor collagenase suitable for use with the invention include, but are notlimited to lyophilized or other dried powder for reconstitution prior toinjection, in multiple or single dose amounts, individual dosage unitsready for injection (which preferably also include one or morepreservatives), frozen unit dosage forms, or any mode of preparationknown in the art. The formulations also may be provided in the form of akit, which can contain the collagenase in solid form, liquid or solventfor reconstitution and injection, and any equipment necessary foradministration, such as a syringe and needle, particularly a specializedsyringe and/or needle for administration to a uterine fibroid.Preferably, the dosage form has a largest dimension between 1 mm and 20mm. Preferably, the formulations are sterile. The products may besterilized by any method known in the art, such as by filtration througha bacterial-retaining filter or are produced under aseptic conditions.Other methods include exposing the formulation or components thereof toheat, radiation or ethylene oxide gas.

Some examples of materials which can serve as pharmaceuticallyacceptable carriers are solvents for injection as known in the art.Examples include, but are not limited to sterile water, bufferingsolutions, saline solutions such as normal saline or Ringer's solution,pyrogen-free water, ethyl alcohol, non-toxic oils, and the like, or anysolvent compatible with injection or other forms of administration asdescribed herein for use with the invention.

In addition, any solid excipients known in the art for use inpharmaceutical products can be used with the invention as a vehicle orfiller, for example. Sugars such as lactose, glucose and sucrose;starches such as corn starch and potato starch; cellulose and itsderivatives such as microcrystalline cellulose, sodium carboxymethylcellulose, ethyl cellulose and cellulose acetate; powdered tragacanth;malt; gelatin; gums; talc; glycols such as propylene glycol; esters suchas ethyl oleate and ethyl laurate; agar, and the like can be used.Buffering agents compatible with the active compounds and the methods ofuse are contemplated for use, including acid or alkali compounds, suchas magnesium hydroxide and aluminum hydroxide, citric acid, phosphate orcarbonate salts and the like. Non-toxic compatible excipients such aslubricants, emulsifiers, wetting agents, suspending agents, binders,disintegrants, preservatives or antibacterial agents, antioxidants,sustained release excipients, coating agents and the like (e.g., sodiumlauryl sulfate and magnesium stearate) also may be used, as well ascoloring agents, perfuming agents, viscosity enhancing agents,bioadhesives, and the like, according to the judgment of the formulator.

For example, one or more biodisintegrable binders may be included in theformulations of the present invention, typically in connection withdosage forms having solid characteristics, wherein the formulation is adosage form having a largest dimension between 1 mm and 20 mm. Whereemployed, a wide range of biodisintegrable binder concentrations may beutilized, with the amounts varying based, for example, on the desiredphysical characteristics of the resulting dosage form and on thecharacteristics of the uterine fibroid treatment agent that is selected(e.g., the degree of dilution, release delay, etc. that isdesired/tolerated), among other considerations. The concentration ofbiodisintegrable binder typically ranges are from about 1 to 80 wt % ofbiodisintegrable binder, more typically about 5 to 50 wt %. A“biodisintegrable” material is one that, once placed in tissue such asuterine tissue, undergoes dissolution, degradation, resorption and/orother disintegration processes. Where such materials are included,formulations in accordance with the present invention will typicallyundergo at least a 10% reduction in weight after residing in tissue suchas uterine tissue for a period of 7 days, more typically a 50-100%reduction in weight after residing in the tissue for a period of 4 days.Suitable biodisintegrable binders for use in connection with the presentinvention include, but are not limited to biodisintegrable organiccompounds, such as glycerine, and biodisintegrable polymers, or anyknown disintegrant compound known in the art of pharmaceutics.

Where used, viscosity adjusting agent(s) are typically present in anamount effective to provide the formulation with the desired viscosity,for example, by rendering the formulation highly viscous, for example,in an amount effective to provide a viscosity between about 5,000 and200,000 centipoise, more typically between about 10,000 and 100,000centipoise, more typically between about 10,000 and 50,000 centipoise,and even more typically between about 20,000 and 40,000 centipoise. Byproviding formulations having viscosities within these ranges, theformulations can be injected into tissue, such as uterine tissue, usingconventional injection equipment (e.g., syringes). However, due to theirelevated viscosities, the formulations have improved retention withinthe tissue at the injection site. The concentration of the viscosityadjusting agent(s) that is (are) used can vary widely. Commonly, theoverall concentration of the viscosity adjusting agent(s) is betweenabout 1 and 20 wt %. In many embodiments, the viscosity adjusting agentsare polymers, which may be of natural or synthetic origin and aretypically biodisintegrable. The polymers are also typically watersoluble and/or hydrophilic. However, in some embodiments, for instancewhere an organic solvent such as dimethylsulfoxide (DMSO) is used as aliquid component, the viscosity adjusting agent can be relativelyhydrophobic. The polymeric viscosity adjusting agents includehomopolymers, copolymers and polymer blends.

Examples of viscosity adjusting agents for the practice of the presentinvention include, but are not limited to the following: cellulosicpolymers and copolymers, for example, cellulose ethers such asmethylcellulose (MC), hydroxyethylcellulose (HEC), hydroxypropylcellulose (HPC), hydroxypropyl methyl cellulose (HPMC),methylhydroxyethylcellulose (MHEC), methylhydroxypropylcellulose (MHPC),carboxymethyl cellulose (CMC) and its various salts, including, e.g.,the sodium salt, hydroxyethylcarboxymethylcellulose (HECMC) and itsvarious salts, carboxymethylhydroxyethylcellulose (CMHEC) and itsvarious salts, other polysaccharides and polysaccharide derivatives suchas starch, hydroxyethyl starch (HES), dextran, dextran derivatives,chitosan, and alginic acid and its various salts, carrageenan, variousgums, including xanthan gum, guar gum, gum arabic, gum karaya, gumghatti, konjac and gum tragacanth, glycosaminoglycans and proteoglycanssuch as hyaluronic acid and its salts, heparin, heparin sulfate,dermatan sulfate, proteins such as gelatin, collagen, albumin, andfibrin, other polymers, for example, carboxyvinyl polymers and theirsalts (e.g., carbomer), polyvinylpyrrolidone (PVP), polyacrylic acid andits salts, polyacrylamide, polyacrylic acid/acrylamide copolymer,polyalkylene oxides such as polyethylene oxide, polypropylene oxide andpoly(ethylene oxide-propylene oxide) (e.g., Pluronic acid),polyoxyethylene (polyethylene glycol), polyethyleneamine andpolypyrridine, poly-metaphosphate (Kurrol salts), polyvinyl alcohol,additional salts and copolymers beyond those specifically set forthabove, and blends of the foregoing (including mixtures of polymerscontaining the same monomers, but having different molecular weights),and so forth. Many of these species are also useful as binders.

In other embodiments of the invention, formulations or carriers arecrosslinked, either prior to use or in vivo. Crosslinking isadvantageous, for example, in that it acts to improve formulationretention (e.g., by providing a more rigid/viscous material and/or byrendering the polymer less soluble in a particular environment). Wherethe formulation is crosslinked in vivo, a crosslinking agent is commonlyinjected into tissue either before or after the injection or insertionof a formulation in accordance with the present invention. Depending onthe nature of the formulation and the crosslinking agent, theformulation may be converted, for example, into a solid, into asemi-solid, or into a high-viscosity fluid.

Crosslinking agents suitable for use in the present invention include,any non-toxic crosslinking agent, including ionic and covalentcrosslinking agents. For example, in some embodiments, polymers areincluded within the formulations of the present invention, which areionically crosslinked, for instance, with polyvalent metal ions.Suitable crosslinking ions include polyvalent cations selected from thegroup consisting of calcium, magnesium, barium, strontium, boron,beryllium, aluminum, iron, copper, cobalt, lead and silver cations ions.Polyvalent anions include phosphate, citrate, borate, succinate,maleate, adipate and oxalate anions. More broadly, crosslinking anionsare commonly derived from polybasic organic or inorganic acids. Ioniccrosslinking may be carried out by methods known in the art, forexample, by contacting ionically crosslinkable polymers with an aqueoussolution containing dissolved ions.

In some embodiments, polymers are included, which are covalentlycrosslinkable, for example, using a polyfunctional crosslinking agentthat is reactive with functional groups in the polymer structure. Thepolyfunctional crosslinking agent can be any compound having at leasttwo functional groups that react with functional groups in the polymer.Various polymers described herein can be both covalently and ionicallycrosslinked.

Suitable polymers for ionic and/or covalent crosslinking can beselected, for example, from the non-limiting list of the following:polyacrylates; poly(acrylic acid); poly(methacrylic acid);polyacrylamides; poly(N-alkylacrylamides); polyalkylene oxides;poly(ethylene oxide); poly(propylene oxide); poly(vinyl alcohol);poly(vinyl aromatics); poly(vinylpyrrolidone); poly(ethylene imine);poly(ethylene amine); polyacrylonitrile; poly(vinyl sulfonic acid);polyamides; poly(L-lysine); hydrophilic polyurethanes; maleic anhydridepolymers; proteins; collagen; cellulosic polymers; methyl cellulose;carboxymethyl cellulose; dextran; carboxymethyl dextran; modifieddextran; alginates; alginic acid; pectinic acid; hyaluronic acid;chitin; pullulan; gelatin; gellan; xanthan; carboxymethyl starch;hydroxyethyl starch; chondroitin sulfate; guar; starch; and salts,copolymers, mixtures and derivatives thereof.

In one preferred embodiment, the collagenase is formulated as alyophilized injectable composition formulated with lactose, sucrose orany suitable sugar. One preferred collagenase composition is alyophilized injectable composition formulated with sucrose, Tris at a pHlevel of about 8.0. Most preferably, 1.0 mg of the drug substance of theinvention is formulated in 60 mM sucrose, 10 mM Tris, at a pH of about8.0 (e.g., about 20.5 mg/mL of sucrose and 1.21 mg/mL of Tris in theformulation buffer).

Preferred collagenase compositions for use in the invention comprise amixture of collagenase I and collagenase II has a specific activity ofat least about 700 SRC units/mg, such as at least about 1000 SRCunits/mg, more preferably at least about 1500 SRC units/mg. One SRC unitwill solubilize rat tail collagen into ninhydrin reaction materialequivalent to 1 nanomole of leucine per minute, at 25° C., pH 7.4.Collagenase has been described in ABC units as well. This potency assayof collagenase is based on the digestion of undenatured collagen (frombovine tendon) at pH 7.2 and 37° C. for 20-24 hours. The number ofpeptide bonds cleaved are measured by reaction with ninhydrin. Aminogroups released by a trypsin digestion control are subtracted. One netABC unit of collagenase will solubilize ninhydrin reactive materialequivalent to 1.09 nanomoles of leucine per minute. One SRC unit equalapproximate 6.3 ABC unit or 18.5 GPA unit. In one embodiment, eachmilligram of collagenase for injection will contain approximately 2800SRC units.

Doses contemplated for administration by direct injection to the uterinefibroid tissue will vary depending on the size of the tissue to betreated and the discretion of the treating physician. However, dosesgenerally are about 0.06 mg collagenase to about 1 mg collagenase percm³ of tissue to be treated or about 0.1 mg collagenase to about 0.8 mgcollagenase per cm³ of tissue to be treated, or about 0.2 mg collagenaseto about 0.6 mg collagenase per cm³ of tissue to be treated.

Formulations that contain an additional active agent or medication alsoare contemplated. Optional additional agents which can be included inthe formulation for concomitant, simultaneous or separate administrationinclude, for example, any pharmaceutical known in the art for shrinkage,treatment or elimination of uterine fibroids or their symptoms, or toassist in performance of the present treatment methods. For example, oneor more fibroid treatment agents such as aromatase inhibitors (e.g.,letrozole, anastrozole, and exemestande), progesterone receptor agonistsand modulators (e.g., progesterone, progestins, mifepristone,levonoergestrel, norgestrel, asoprisnil, ulipristal and ulipristalacetate, telepristone), selective estrogen receptor modulators (SERMs)(e.g., benzopyran, benzothiophenes, chromane, indoles, naphtalenes,tri-phenylethylene compounds, arzoxifene, EM-652, CP 336,156,raloxifene, 4-hydroxytamoxifen and tamoxifen), gonadotrophin-releasinghormone analogs (GnRHa) (e.g., GnRH agonist peptides or analogs withD-amino acid alterations in position 6 and/or ethyl-amide substitutionsfor carboxyl-terminal Gly10-amide such as triptorelin or GnRHantagonists such as cetrorelix, ganirelix, degarelix and ozarelix),growth factor modulators (e.g., TGFb neutralizing antibodies),leuprolide acetate, non-steroidal anti-inflammatory drugs, inhibitors ofthe mTOR pathway, inhibitors of the WNT signaling pathway, vitamin D,vitamin D metabolites, vitamin D modulators, and/or an additionalanti-fibrotic compound (e.g., pirfenidone and halofuginone) may beco-administered with collagenase in the same or a separateadministration.

Chemical ablation agents also can be included in the formulations of thepresent invention. In effective amounts, such compounds cause tissuenecrosis or shrinkage upon exposure. Any known ablation agent can beused according to the art, in concentrations as appropriate to theconditions while avoiding inactivation of the collagenase, with theamounts employed being readily determined by those of ordinary skill inthe art. Typical concentration ranges are from about 1 to 95 wt % ofablation agent, more typically about 5 to 80 wt %. Ablation agentssuitable for use with the invention include, but are not limited toosmotic-stress-generating agents (e.g., a salt, such as sodium chlorideor potassium chloride), organic compounds (e.g., ethanol), basic agents(e.g., sodium hydroxide and potassium hydroxide), acidic agents (e.g.,acetic acid and formic acid), enzymes (e.g., hyaluronidase, pronase, andpapain), free-radical generating agents (e.g., hydrogen peroxide andpotassium peroxide), oxidizing agents (e.g., sodium hypochlorite,hydrogen peroxide and potassium peroxide), tissue fixing agents (e.g.,formaldehyde, acetaldehyde or glutaraldehyde), and/or coagulants (e.g.,gengpin). These agents may be combined with collagenase in the sameformulation so long as they do not negatively affect the enzymaticactivity of the collagenase, or they may be administered separately, atthe same time or at different times.

The methods according to the invention may be used in conjunction withany known treatments to control symptoms caused by fibroids. Forexample, NSAIDs or other analgesics can be used to reduce painfulmenses, oral contraceptive pills are may be prescribed to reduce uterinebleeding, and iron supplementation may be given to treat anemia. Alevonorgestrel intrauterine device can be used to reduce hemorrhage andother symptoms if the condition of the uterus does not result inexpulsion of the device.

The ability to non-invasively image regions where the formulations ofthe present invention are being introduced and where they have beenintroduced is a valuable diagnostic tool for the practice of the presentinvention. Therefore, in addition to a uterine fibroid treatment agentand any of the various optional components discussed above, the uterinefibroid formulations of the present invention also optionally includeone or more imaging contrast agents to assist with guiding the clinicianto administer the collagenase compound to the fibroid or tissue to betreated or to determine that administration has been correctly located.Non-non-invasive imaging techniques include magnetic resonance imaging(MRI), ultrasonic imaging, x-ray fluoroscopy, nuclear medicine, andothers. Any contrast agent suitable for use with such techniques andknown in the art can be used as part of the inventive compositions andformulations.

Any real-time imaging technology can be used to guide injection orinsertion in the invention. For example, X-ray based fluoroscopy is adiagnostic imaging technique that allows real-time patient monitoring ofmotion within a patient. To be fluoroscopically visible, formulationsare typically rendered more X-ray absorptive than the surroundingtissue. In various embodiments of the invention, this is accomplished bythe use of contrast agents. Examples of contrast agents for use inconnection with X-ray fluoroscopy include metals, metal salts and oxides(particularly bismuth salts and oxides), and iodinated compounds. Morespecific examples of such contrast agents include tungsten, platinum,tantalum, iridium, gold, or other dense metal, barium sulfate, bismuthsubcarbonate, bismuth trioxide, bismuth oxychloride, metrizamide,iopamidol, iothalamate sodium, iodomide sodium, and meglumine.

Ultrasound and magnetic resonance imaging can provide two- and/orthree-dimensional images of a portion of the body. Ultrasound and MRIare advantageous, inter alia, because they do not expose the patient ormedical practitioner to harmful radiation and they can provide detailedimages of the observed area. These detailed images are valuablediagnostic aids to medical practitioners and can be used to moreprecisely control the quantity and location of the formulations of thepresent invention.

Suitable ultrasonic imaging contrast agents for use in connection withthe present invention include solid particles ranging from about 0.01 to50 microns in largest dimension (e.g., the diameter, where sphericalparticles are used), more typically about 0.5 to 20 microns. Bothinorganic and organic particles can be used. Examples includemicroparticles/microspheres of calcium carbonate, hydroxyapatite,silica, poly(lactic acid), and poly(glycolic acid). Microbubbles canalso be used as ultrasonic imaging contrast agents, as is known in theimaging art. The ultrasonic imaging contrast agents for use inconnection with the present invention are preferably biocompatible andstable in the formulation. Concentrations of the ultrasonic imagingcontrast agents typically range from about 0.01 wt % to 10 wt % of theformulation, more typically about 0.05 to 2 wt %, where solid particlesare used.

For contrast-enhanced MRI, a suitable contrast agent has a largemagnetic moment, with a relatively long electronic relaxation time.Based upon these criteria, contrast agents such as Gd(III), Mn(II) andFe(III) can be used. Gadolinium(III) has the largest magnetic momentamong these three and is, therefore, a widely-used paramagnetic speciesto enhance contrast in MRI. Chelates of paramagnetic ions such asGd-DTPA (gadolinium ion chelated with the liganddiethylenetriaminepentaacetic acid) also are suitable. Furtherinformation can be found, for example, in U.S. Patent Application No.2003-0100830 entitled “Implantable or insertable medical devices visibleunder magnetic resonance imaging,” the disclosure of which isincorporated herein by reference.

The collagenase formulations described here preferably are injected intoone or more individual uterine fibroid tumors using a hollow deliverychannel, such as a hollow needle or cannula. For instance,administration can be performed using a needle in association with aconventional or specially designed syringe, cannula, catheter, and thelike. A source of manual, mechanical, hydraulic, pneumatic or othermeans to apply pressure (e.g., a conventional syringe plunger, a pump,aerosol, etc.) can be used to inject the formulation into the fibroid.Alternatively, the formulations can be administered during surgery, forexample via a trocar during laparoscopic surgery and duringhysteroscopic treatment.

Injection routes include, for example, transabdominal, transcervical andtransvaginal routes. Where the formulations have fluid attributes, theinjection volume will vary, depending, for example, on the size of thefibroid, the type and concentration of treatment agent, and so forth,and will typically range from 1.0 to 10.0 ml per injection. Similarly,where formulations having solid attributes (e.g., pellets or powders)are used, the amount of formulation injected/inserted will also depend,for example, on the size of the fibroid, the type and concentrationtreatment agent utilized, etc. Multiple pellets or doses of collagenasecomposition can be administered at a single injection site. Regardlessof the physical attributes of the formulation, multipleinjection/insertion sites may be established within a single fibroid,with the number of injections depending on the size and shape of thefibroid as well as the type and/or concentration of the treatment agentthat is used. Multiple fibroids or a single fibroid can be treated.

In various embodiments, the injection/insertion device is guided to thefibroid site under image guidance. Image guidance can include, forexample, direct visual guidance (e.g., laparoscopic guidance intrans-abdominal procedures and hysteroscopic guidance in trans-vaginalprocedures) and non-direct visual guidance (e.g., ultrasound guidance,fluoroscopic guidance, and/or MRI guidance).

As a specific example, visual guidance of the injection/insertion deviceis conducted laparoscopically using a scope that is positioned in theabdomen (e.g., by insertion through a trocar). In this way, a device(e.g., a delivery needle or canula) can be inserted percutaneously intothe abdomen and guided under laparoscopic vision to the uterine fibroid.Once the fibroid is reached, fluoroscopy, MRI or ultrasound (e.g.,trans-vaginal ultrasound, trans-abdominal ultrasound, intra-abdominalultrasound, etc.) preferably is used to guide the tip of the deliveryneedle to a desired position within the fibroid, at which point theformulation is injected or inserted into the fibroid. To the extent thatthere is sufficient contrast between the formulation and the surroundingtissue, the location of the formulation within the fibroid will also beviewed.

The compositions and processes of the present invention will be betterunderstood in connection with the following examples, which are intendedas an illustration only and not limiting of the scope of the invention.Various changes and modifications to the disclosed embodiments will beapparent to those skilled in the art and such changes and modificationsincluding, without limitation, those relating to the processes,formulations and/or methods of the invention may be made withoutdeparting from the spirit of the invention and the scope of the appendedclaims.

EXAMPLES Example 1. General Collagenase Production

To prepare an animal-material-free clostridia cell bank, Clostridiumhistolyticum cells are suspended in a medium containing a vegetablepeptone and optionally yeast extract. For example, one general methodfor accomplishing this is as follows.

TABLE 2 General Method to Produce Clostridium Cell Bank. Step 1 Startingcells: any Clostridium histolyticum culture which is convenient andavailable, for example Clostridium histolyticum ATCC 21000, strain 004Step 2 Inoculate 1 mL of step 1 into 300 mL of media containing 15.45 gPhytone, 2.55 g yeast extract, and water sufficient to produce 0.3 L(M#1); step 2 for 24 hours at 37° C. (1^(st) culture); Step 4 Transfer 3mL of step 3 (1^(st) culture) to 1000 mL of M#1; Step 5 Incubate step 4for 16 hours at 37° C. (2^(nd) culture); Step 6 Centrifuge the 2^(nd)culture; Step 7 Re-suspend the pellet with the 5 mL of media #1 and 5 mLof 20% glycerol; Step 8 Freeze the aliquot of cells gradually; Step 9Store the aliquot at −80° C.

Once an animal material-free cell bank is established, the cells can begrown or fermented in convenient media known in the art, preferablynon-animal-derived medium. The medium can optionally contain yeastextract. Exemplary, non-limiting examples of such media are M#1, M#2,M#3, and M#4 as described in Table 3, below. In addition, see Table 4for an exemplary, non-limiting general example of the steps of thefermentation process.

TABLE 3 Media recipes and preparation. M #1 M #2 M #3 M #4 Phytone 15.45g 103 g Veggitone 15.45 g 103 g Yeast extract 2.55 g 2.55 g 17 g 17 gKH₂PO₄ 1.92 g 1.92 g K₂HPO₄ 1.25 g 1.25 g Na₂HPO₄ 3.5 g 3.5 g NaCl 2.5 g2.5 g vol of water 0.3 L 0.3 L 1 L 1 L

TABLE 4 Fermentation Process. Step 1 Starting cells: Animal materialfree clostridia cell bank Step 2 Inoculate 1 mL of step 1 into the 300mL of M#1; Step 3 Incubate step 2 for 16 to 24 hours at 37° C. (1^(st)culture); Step 4 Transfer 10 mL of step 3 (1^(st) culture) and 10 mLVitamin/Mg solution* to 1000 mL of M#3, or 4 respectively; Step 5Incubate step 4 for about 22 hours at 37° C. (2^(nd) culture); Step 6Use 2^(nd) culture for downstream isolation and purification. *Preparedseparately by dissolving 8 g MgSO₄, 1.2 g ferrous sulfate, 0.05 griboflavin, 0.1 g Niacin, 0.1 g Calcium pantothenate, 0.1 g pimelicacid, 0.1 g pyridoxine, and 0.1 g thiamine in 1100 mL water, followed bysterilization by 0.22 um filtration.

After preparation of “2^(nd) culture,” the collagenase I and collagenaseII can be isolated and purified using any method capable of producingeach enzyme separately to at least 95% purity. The method may combineone or more of the steps of ammonium sulfate precipitation, dialysis,hydroxyapatite (HA) chromatography, gel filtration and ion-exchange, forexample, preferably in that order. The gel filtration is preferably G75gel filtration. The ion-exchange is preferably anion-exchange:Q-Sepharose chromatography. In addition, when the Clostridia have beencultured in medium containing less glucose and more salt compared to themajority of known bacterial culture, as preferred, protease inhibitorssuch as leupeptin are not required.

Example 2. Preparation of Animal Material Free Clostridium Cell Bank

The starter cell culture was Clostridium histolyticum ATCC 21000, strain004 which was originally created with bovine-derived materials. Thecells were first grown in animal material free medium (M #1, Table 3).Briefly, the recipe includes: phytone, 51.5 g, yeast extract 8.5 g, 1000mL water. The pH was adjusted to 7.30 with NaOH, and the mediumsterilized at 121° C. for 20 minutes. One milliliter of the startingmaterial was then inoculated into 300 mL of M#1 and incubated for 24hours at 37° C. (1st culture). Three milliliters of the 1st culture wastransferred to 1000 mL of M#1 and incubated for 16 hours (2nd culture).The 2nd culture was then centrifuged aseptically. The pellet wasre-suspended in 5 mL M#1 with 5 mL 20% glycerol. The aliquots of cellsuspension were frozen gradually and stored at −80° C.

Example 3. Fermentation Process

Clostridium histolyticum ATCC 21000, strain 004 was inoculated into thestarting culture with M#1 or M#2 and incubated at 37° C. for 16 hours.Ten milliliters of the starting culture (M#1 or M#2) and 10 mLMg/vitamin solution (prepared separately by dissolving 8 g MgSO4, 1.2 gferrous sulfate, 0.05 g riboflavin, 0.1 g Niacin, 0.1 g Calciumpantothenate, 0.1 g pimelic acid, 0.1 g pyridoxine, and 0.1 g thiaminein 1100 mL water, followed by sterilization by 0.22 μm filtration) wasthen transferred to each liter of M#3 or M#4 (or a variation thereof),and incubated for 22 hours. Clostridium histolyticum grew well with theOD600 reaching >2.5.

Example 4. General Procedure for Isolation and Purification ofCollagenase I and Collagenase II

TABLE 5 General Exemplary, Non-Limiting Isolation and PurificationProcedure for Collagenase I and Collagenase II. Stages of ProductOperations Fermentation Centrifugation or 1.0 μm filtration; brothClarified Add ammonium sulfate (590 g/liter); centrifugation;fermentation broth Crude Dissolve crude collagenase precipitate byadding collagenase purified water; precipitate Crude collagenase Dialyzecrude collagenase solution against purified solution (store waterovernight with 10 kDa pore size dialysis at −20° C.) membrane; Dialyzedcrude Clarify the dialyzed crude collagenase solution with collagenaseeither centrifugation or filtration or the combination of both;Clarified Add potassium phosphate buffer, pH 6.7 to a final solutionconc. of 0.1M; Collagenase in Load collagenase solution tohydroxylapatite column phosphate buffer and elute column with gradientof increasing K₂PO₄ conc. at ambient temp. (20° C.); CollagenaseConcentrate the eluate with ultrafiltration (30 kDa of HA eluate poresize); Concentrated Load the concentrate onto a G75 gel filtrationcolumn collagenase at ambient temperature (20° C.) and elute with 20 mMTris/150 mM NaCl; Collagenase Dialyze the eluate against a buffer (10 mMTris, 3 mM G75 eluate calcium chloride (CaCl₂), pH 8.0) overnight;Dialyzed Load dialyzed eluate on to a Q-Sepharose anion- G75 eluateexchange column at ambient temperature (20° C.); elute using a gradientof 10 mM Tris HCl, 3 mM CaCl₂, pH 8.0 buffer and 10 mM Tris HCl, 3 mMCaCl₂, 1M NaCl, pH 8.0 buffer; Collagenase Store separately at −20° C.class I and class II fractions

Example 5. Ex Vivo Treatment of Uterine Fibroid Tissue

Samples of fibroid tissue and myometrium were obtained post-hysterectomyfrom women with consent and identified by evaluation by a surgicalpathologist. The tissue samples were transported to the laboratory andcut into 1 cm³ cubes. See FIG. 2. These cubes were injected withpurified collagenase (0.06 or 0.2 mg in 100 μL) dissolved in media orserum and then incubated for 24, 48, 72, or 96 hours at 37° C. See FIG.3. Each treatment was carried out in tissues from three differentpatients with two tissue samples per treatment because fibroid tissue isextremely variable. Control fibroid and myometrium cubes were injectedwith vehicle or sham injected. At the end of the incubation, the tissuesamples were photographed to document gross appearance. Degree ofliquefaction and softening was observed and documented using a 4-pointsubjective scale.

Samples were frozen for biomechanical assessment (compression analysis).Samples were fixed in formalin for histology and Masson trichrome andpicrosirius red staining. They were analyzed by light microscopy for thepresence or absence of collagen and assessed using computer morphometryto determine the extent of degradation. In the case of picrosirius redstaining, polarized light microscopy was performed to determine collagenfiber orientation. Samples were fixed in glutaraldehyde and postfixedwith osmium tetraoxide for electron microscopy to determine collagenfibril orientation and evidence of fibril degradation. Additionalinjections were done at a dose of 0.58 mg/injection (250 ul of 2.3mg/ml).

These ex-vivo studies have shown the efficacy of purified collagenase insoftening and partial liquefaction of post-hysterectomy fibroidspecimens, as well as a decrease in the collagen content. Treatedfibroid-specimens were grossly softer and had partially liquefiedcenters. Masson trichrome and picrosirius red stains of theses tissuesshowed a dramatic subjective decrease in collagen content compared tofibroid tissue injected with vehicle.

Example 6. Treatment of Whole Uterine Fibroids Ex Vivo

Donated tissue was obtained from four female adult patients 18 years ofage or older who can give legally effective consent and who wereplanning to undergo definitive treatment for fibroids by hysterectomy.After the removal of the hysterectomy specimen, the uterus was observedgrossly by standard procedures by a surgical pathologist. Completefibroids (submucosal (abutting the endometrium), intramural (within themyometrium), and subserosal (abutting the uterine serosa) fibroids, orpedunculated fibroids (attached to the uterus by a stalk) if they arepresent) from 1 to 4 cm (including the capsule) along with 1.5 cm of thesurrounding adjacent myometrium and, if available, a 0.5 cm section ofendometrium were dissected free from the specimen and placed in normalsaline.

Tissues were brought to the laboratory immediately, washed and injectedwith purified Clostridium histolyticum collagenase (PCHC) (0.1 mg/100μl/cm³). Optionally, a higher concentration of the collagenase was usedto decrease the volume of the injection. Purified collagenase wasdiluted in 0.3 mg/mL calcium chloride dihydrate in 0.9% sodium chloride,optionally combined with 1% methylene blue as a marker to visuallyassess the area of distribution of the injected material within thefibroid and uterus. Fibroids were injected with PCHC or vehicle in thecenter of the obtained specimen. See FIGS. 4A and 4B. The amount ofcollagenase injected depended on the size of the fibroids (1-4 cm).Generally, about 818 μL of material was injected into a fibroid with adiameter of about 2.5 cm. If injecting the entire treatment volumecentrally was not feasible due to tissue resistance to the injection orother factors, multiple locations were injected within the fibroid. Thefibroid tissue then was incubated in DMEM/F12 culture medium at 37° C.for 24 hours. At least one fibroid with attached myometrium served asthe control. This specimen received an injection of 1% methylene blue invehicle without collagenase as a non-randomized placebo injection,centrally into the fibroid.

Color photographs were taken of the uterus and of the fibroid andmyometrial pieces pre- and post-injection. Fibroid diameters weremeasured with a metric ruler.

At the end of the incubation, the samples were reassessed grossly forsize, consistency and firmness, and color photographs were obtained, aswell as optional video recording to record fibroid manual distensibilityand any liquefied portions upon sectioning. The degree of liquefactionand softening were observed and documented using a 4-point subjectivescale.

Whether the collagenase can penetrate the capsule and affect the nearbymyometrium was determined. Samples were obtained, including tissue fromthe injected fibroid and adjacent tissue, plus a section that includedfibroid and adjacent myometrium and/or endometrium still attached, andmyometrium alone. Samples were fixed in formalin for histology andMasson trichrome, picrosirius red, and hematoxylin-eosin staining. Thesamples were analyzed by light microscopy for the presence or absence ofcollagen and using computer morphometry to assess the extent ofdegradation. Picrosirius red staining was used with polarized lightmicroscopy to determine collagen fiber orientation.

Exemplary treatment schemes for each patient:

fibroid 1: inject 818 μL 1 mg/mL collagenase;

fibroid 2: inject 818 μL 1 mg/mL collagenase;

fibroid 3: inject 818 μL control vehicle;

Injections were given through the fibroid capsule into the center of thefibroid, through the myometrium into the center of the fibroid, orthrough the endometrium into the center of the fibroid, simulating invivo injection routes. The fibroids here were liquefied in the samemanner as shown in FIG. 5 (see below).

Example 7. Biomechanical Evaluation of Human Uterine Fibroids afterInjection with Purified Clostridial Collagenase

The two collagenases isolated from Clostridium histolyticum (ABC I andABC II) were combined in a 1:1 mass ratio. Both collagenases aremetalloproteases and have a broad hydrolyzing reactivity and degradetype I and Ill collagens. The biomechanical properties of uterinefibroid tissue were analyzed by rheometry in control andcollagenase-treated specimens.

Uterine fibroids have been shown to contain about 70% Type I collagencompared to about 80% in myometrium; about 28% Type III collagencompared to about 20% in myometrium; and about 5% Type V collagencompared to about 2% in myometrium. Type Willis lower at the center andthe edge of fibroids as compared to myometrium. (Feng et al,

Fibroid tissue was obtained after surgery (hysterectomy or myomectomy)from 4 different patients and cut into cubes (1 cm³; n=43). Tissue cubeswere injected into the center with 100 μL of purified collagenase (0,0.25, 0.5, 1.0, 2.0 mg/mL; n=4-14 per dose) and incubated at 37° C. for24, 48, or 96 hours. At the end of the incubation period, cubes were cutin half and snap-frozen in liquid nitrogen. Different degrees ofsoftening and liquefaction at the center were noted. An AR-G2 rheometerwas used to measure the sample stiffness dynamically (complex shearmodulus (Pa) at 10 rad/sec), taking into account both the viscous andelastic behavior of the material. At least 2 specimens (5 mm diameterpunch) from each tissue cube were measured. Data were analyzed by 2-wayANOVA and Dunnett's multiple comparisons test.

Overall, stiffness in control fibroid cubes (6585±707 Pa; n=13) wasgreater than in treated cubes (2003±275 Pa; n=30; p<0.0001). Morespecifically, stiffness in fibroid tissues was reduced in a time anddose dependent manner. At 48 hours, treatment with 0.25 mg/mL did notreduce stiffness (5032±1796 Pa), but treatment with 0.5 mg/mL did(2014±1331 Pa; p≤0.05). At 96 hours, both the 0.25 and the 0.5 doseswere effective (1720±377 and 1072±160 Pa; p≤0.01). The 1.0 and 2.0 mg/mLtreatments reduced stiffness at 24 hours, but not significantly (2177±37and 2480±984 Pa; n=4). However, doses of 1.0 and 2.0 mg/mL wereeffective at 48 hours (3588±637; p≤0.05 and 1254±445 Pa; p≤0.01; n=6)and at 96 hours (921±305 and 1350±571 Pa; p≤0.0001; n=10).

Using a torsional rheometer, tissue stiffness was quantitated over awide range (very firm to liquefied). Our data indicate that treatment ofthe fibroid tissue with defined doses of purified clostridialcollagenase significantly decreased the stiffness (modulus) of thetissue. See FIG. 5, which shows collagenolysis in fibroid tissue after48 hour incubation. The left photograph is tissue that was injected withvehicle (control) and the right photograph is tissue that was injectedwith collagenase. FIG. 6 shows micrographs of control (FIGS. 6A and 6B)and collagenase-treated (FIGS. 6C and 6D) tissue. Mason stain in FiguresA and C (left) shows that collagen is decreased. Picrosirus red stainvisualized under polarized light (FIG. 6D) clearly shows in the bottomright that collagen fibers are degraded.

Example 8. Treatment of Human Uterine Fibroids in Nude Mouse Model

The xenograft mouse model, in which three-dimensional organotypiccultures of human uterine fibroid cells are implanted under the skin offemale nude mice, has been successfully employed to study keloids, afibrotic skin disorder with biology similar to fibroids. This model isused to demonstrate effects of PCHC injection, in an HPG nanocarrierformulation, on fibroid tissue in vivo.

Polylactic acid sponges, other synthetic polylactic acid scaffolds, orany suitable commercially available scaffold is inoculated with humanuterine fibroid cells to produce an organotypic 3-D culture of uterinefibroid cells that can be implanted into nude mice. These 3-dimensionalorganotypic cultures (3D-fibroids) are representative of human fibroidsand produce and contain extracellular matrix.

OPLA sponges (Open-Cell Polylactic Acid, BD Biosciences; FIG. 7) aresynthetic polymer scaffolds that are synthesized from D,D-L,L polylacticacid. This material has a facetted architecture which is effective forculturing high density cell suspensions. The cells will be seeded ontothe 3D sponge-like scaffolds under dynamic conditions, leading touniform cell population throughout the sponges and higher cell numbersper sponge than static seeding. Post-sterilization, the molecular weightof the OPLA is 100-135 kD. They have an approximate size of 5 mm×3 mm(0.04 cm³) with an average pore size of 100-200 μm.

Cells and scaffolds are placed into cell culture chambers of abioreactor consisting of a fluid (culture media)-filled, rotatingchamber that allows for constant floating of cells while minimizingshearing forces and gravitational settlement of cells and/or scaffolds(Synthecon, Inc.). Cells inside the rotating bioreactor chamber aresuspended in virtual weightlessness.

Primary human fibroid cells from specimens obtained at hysterectomy areseeded statically or dynamically into OPLA sponges and grown for 30 daysto allow for production and assembly of extracellular matrix. Cells growthroughout the scaffold and can be formalin fixed, paraffin embedded andthin sectioned for observation, optionally with staining for multiplemarkers. See FIG. 8, which shows the formation of the cell latticefollowing the outlines of the sponge-like scaffold.

FIG. 9 shows primary cultures of fibroid cells after static seeding. Thecells are fixed on the scaffold and observed in situ. Scaffoldscontaining cells were fixed and were unstained (FIG. 9A) or stained forf-actin with fluorescent phalloidin (FIG. 9B). Cells were evenlydistributed throughout the scaffold. The imaged scaffolds are >1 mmthick and therefore not all cells are in focus, indicating that thecells are growing not only on the surface, but also deep inside thescaffolds. FIG. 10 shows the population of cells throughout thesponge-like scaffolds using confocal microscopy (FIGS. 10A and 10B).

High quality RNA is extracted from the 3D-cultures of fibroid cells onOPLA sponges and used to verify the expression of two genes of interest.Versican and TGFβ3 are known to be highly expressed in fibroid tissueand cells. Results in Table 6 show that both a fibroid cell line andprimary cultures of fibroid cells in this 3D-culture system expressthese two genes in high amounts.

TABLE 6 Real Time PCR Assay Results cDNA (ng) per Threshold Cycle Ct(mean ± SEM) reaction Versican TGFβ₃ Fibroid 50 22.1 ± 0.07 26.8 ± 0.07Cell Line Primary 25 22.2 ± 0.21 24.0 ± 0.04 Fibroid Cells

The patent and scientific literature referred to herein establishes theknowledge that is available to those with skill in the art. All UnitedStates patents and published or unpublished United States patentapplications cited herein are incorporated by reference in theirentireties for all purposes. All published foreign patents and patentapplications cited herein are hereby incorporated by reference in theirentireties for all purposes. All other published references, documents,manuscripts and scientific literature cited herein are herebyincorporated by reference in their entireties for all purposes.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

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The invention claimed is:
 1. A method for the treatment of uterinefibroids in a patient comprising administering into the uterine fibroida composition comprising an amount of collagenase from Clostridiumhistolyticum sufficient to cleave disordered collagen fibrils of typesI, III and V in said uterine fibroids, wherein said administering is byinjecting or inserting into the fibroid about 0.06 to about 1 mg of saidcollagenase per 1 cm³ uterine fibroid tissue.
 2. The method of claim 1,wherein the collagenase is a mixture of collagenase I and collagenaseII.
 3. The method of claim 1, wherein about 0.1 mg to about 0.8 mgcollagenase is administered per cm³ of tissue to be treated.
 4. Themethod of claim 1, wherein about 0.2 mg to about 0.6 mg collagenase isadministered per cm³ of tissue to be treated.
 5. The method of claim 1,wherein said composition is injected or inserted into said fibroidtransabdominally.
 6. The method of claim 1, wherein said composition isinjected or inserted into said fibroid transvaginally.
 7. The method ofclaim 1, wherein said composition is injected or inserted into saidfibroid under image guidance.
 8. The method of claim 7, wherein saidimage is at least one of a direct visual image and a non-direct visualimage.
 9. The method of claim 8, wherein said direct visual image is ascope image.
 10. The method of claim 8, wherein said non-direct visualimage is an MRI image.
 11. The method of claim 10, wherein saidcomposition comprises an MRI contrast agent.
 12. The method of claim 8,wherein said non-direct visual image is an ultrasound image.
 13. Themethod of claim 12, wherein said composition comprises an ultrasoundcontrast agent.
 14. The method of claim 8, wherein said non-directvisual image is a fluoroscopic image.
 15. The method of claim 14,wherein said composition comprises an x-ray contrast agent.
 16. Themethod of claim 1, wherein said composition further comprises a chemicalablation agent, a non-steroidal anti-inflammatory drug, an oralcontraceptive, a GnRH agonist, an antiprogestogen, or a selectiveprogesterone receptor modulator.
 17. The method of claim 16, whereinsaid composition comprises a chemical ablation agent.
 18. The method ofclaim 17, wherein said chemical ablation agent is a salt.
 19. The methodof claim 17, wherein said chemical ablation agent is selected from anenzyme, an acid, a base and an oxidizing agent.
 20. The method of claim1, wherein said composition comprises one or more uterine fibroidtreatment agents.
 21. The method of claim 1, wherein said composition isa solid dosage form having a largest dimension between 1 mm and 20 mm.22. The method of claim 1, wherein said composition is delivered througha delivery channel into said fibroid, wherein the delivery channel is ina needle, syringe, cannula, catheter or jet injector.
 23. The method ofclaim 1, wherein said composition is encapsulated.
 24. The method ofclaim 1, wherein said composition is a powder.
 25. The method of claim24, wherein said powder is introduced into said fibroid by jetinjection.
 26. The method of claim 1, wherein the composition furthercomprises a viscosity adjusting agent is present in an amount effectiveto provide a viscosity ranging from 10,000 centipoise to 50,000centipoise.
 27. The method of claim 1, wherein said composition is afluid formulation that is crosslinked in vivo.
 28. The method of claim1, wherein said composition comprises an alginate polymer.
 29. Themethod of claim 1, wherein said composition comprises gelatin.
 30. Themethod of claim 1, wherein the composition provides sustained release ofan amount of said collagenase sufficient to treat said uterine fibroids.31. The method of claim 1, wherein 100 μL of said composition isinjected or inserted into 1 cm³ uterine fibroid tissue.
 32. A method forthe treatment of uterine fibroids in a patient, comprising administeringinto the uterine fibroid a composition comprising an amount ofcollagenase from Clostridium histolyticum sufficient to cleavedisordered collagen fibrils of types I, III and V in said uterinefibroids, wherein said amount is 0.25-2.0 mg/mL collagenase fromClostridium histolyticum, and wherein said administering is by injectingor inserting said composition into the uterine fibroid tissue.
 33. Themethod of claim 32, wherein said composition comprises 0.25 mg/mLcollagenase from Clostridium histolyticum.
 34. The method of claim 32,wherein said composition comprises 0.5 mg/mL collagenase fromClostridium histolyticum.
 35. The method of claim 32, wherein saidcomposition comprises 1.0 mg/mL collagenase from Clostridiumhistolyticum.
 36. The method of claim 32, wherein said compositioncomprises 2.0 mg/mL collagenase from Clostridium histolyticum.